Epithelial cell hyperplasia in response to an inflammatory response is accompanied by mucous cell metaplasia (MCM), the appearance of mucous cells in airways that are normally devoid of these cells. MCM is one of the major factors for increased mucous hypersecretion in chronic bronchitis. We have shown that Bcl-2, an inhibitor of apoptosis, sustains MCM in rats and mice and may have clinical significance because it is expressed in mucous cells of patients with cystic fibrosis or chronic bronchitis. A polymorphism in the p53 gene at codon 72 (Arg/Pro) differentially affects Bcl-2 expression; p53Pro increases more efficiently than p53Arg, the pro-apoptotic protein Noxa that reduces the bcl-2 mRNA half-life. Deleting or mutating the proline residue within the corresponding region of murine p53 had the same effect in mouse embryo fibroblasts. Consistent with these findings, smokers with the p53Pro/Pro genotype were at lower risk of developing chronic bronchitis than smokers with the p53Arg/Arg genotype. Preliminary results with normal human bronchial epithelial cells (NHBECs) from five Arg/Arg and five Pro/Pro individuals showed that in the untreated state NHBECArg/Arg cultures showed reduced Noxa mRNA levels compared to NHBECPro/Pro cultures and reduced Puma mRNA levels when cultures were treated with environmental cigarette smoke (ETS). Therefore, we want to test the central hypothesis that bronchial cells with the p53Pro/Pro variant resolve epithelial cell hyperplasia and MCM more efficiently than bronchial cells with p53Arg/Arg by inducing Noxa expression that mediates the reduction of Bcl- 2 levels. Specific Aim 1 will determine whether differentiated NHBECArg/Arg, NHBECArg/Pro, and NHBECPro/Pro cultures will show differences in secreted mucus, MUC5AC, Noxa, and Puma mRNA expression, and the development of MCM before and after exposure to ETS extract. Similarly, mouse tracheal epithelial cells from mice with wild-type and mutated p53 will be tested for secreted mucus, and expression of Muc5ac, Noxa and Puma mRNAs, and MCM in the untreated state and after treatment with ETS extract. In addition, five representative NHBECArg/Arg and NHBECPro/Pro cultures will be infected with adenoviral vectors expressing Ad-p53Pro or Ad-p53Arg, respectively, to determine whether the phenotypes for these NHBECs will be modulated. Specific Aim 2 will increase and reduce Noxa and/or Puma expression in NHBECPro/Pro and NHBECArg/Arg cultures to determine their roles in the development of MCM. In addition, the role of these proteins will be examined by exposing noxa-/-, puma-/- and wild-type mice to ETS and by treating NHBECArg/Arg cultures with Noxa- and/or Puma-derived peptides. These studies will provide strategies for novel therapies that may facilitate the restoration of the aberrant repair process and reduce epithelial cell hyperplasia and MCM in chronic bronchitis.